The present invention relates to humanised antibody molecules (HAMs) having specificity for human milk fat globule (HMFG) and to processes for their production using recombinant DNA technology.
The term xe2x80x9chumanised antibody moleculexe2x80x9d (HAM) is used to describe a molecule having an antigen binding site derived from an immunoglobulin from a non-human species, the remaining immunoglobulin-derived parts of the molecule being derived from a human immunoglobulin. The antigen binding site may comprise: either a complete variable domain from the non-human immunoglobulin fused onto one or more human constant domains; or one or more of the complementarity determining regions (CDRs) grafted onto appropriate human framework regions in the variable domain. The abbreviation xe2x80x9cMAbxe2x80x9d is used to indicate a monoclonal antibody.
In the description, reference is made to publications by number. These numbers are placed in square brackets [ ]. The publications are listed in numerical order at the end of the description.
Natural immunoglobulins have been known for many years, as have the various fragments thereof, such as the Fab, Fabxe2x80x2 (Fabxe2x80x2)2 and Fc fragments, which can be derived by enzymatic cleavage. Natural immunoglobulins comprise a generally Y-shaped molecule having an antigen-binding site towards the outer end of each arm. The remainder of the structure, and particularly the stem of the Y, mediates the effector functions associated with immunoglobulins.
Natural immunoglobulins have been used in assay, diagnosis and, to a more limited extent, therapy. However, such uses, especially in therapy, have been hindered by the polyclonal nature of natural immunoglobulins. A significant step towards the realisation of the potential of immunoglobulins as therapeutic agents was the discovery of procedures for the production of monoclonal antibodies of defined specificity [1]. However, most MAbs are produced by hybridomas which are fusions of rodent spleen cells with rodent myeloma cells. The resultant MAbs are therefore essentially rodent proteins. There are few reports of the production of human MAbs.
Since most available MAbs are of rodent origin, they are naturally antigenic in humans and thus can give rise to an undesirable immune response termed the HAMA (Human Anti-Mouse Antibody) response. Therefore, the use of rodent MAbs as therapeutic agents in humans is inherently limited by the fact that the human subject will mount an immunological response to the MAb and will either remove it entirely or at least reduce its effectiveness.
Therefore proposals have been made for making non-human MAbs less antigenic in humans. Such techniques can be generically termed xe2x80x9chumanisationxe2x80x9d techniques. These techniques generally involve the use of recombinant DNA technology to manipulate DNA sequences encoding the polypeptide chains of the antibody molecule.
Early methods for humanising MAbs related to production of chimeric antibodies in which an antigen binding site comprising the complete variable domains of one antibody are fused to constant domains derived from a second antibody. Methods for carrying out such chimerisation procedures are described in EP-A-0 120 694 (Celltech Limited), EP-A-0 125 023 (Genentech Inc.), EP-A-0 171 496 (Res. Dev. Corp. Japan), EP-A-0173494 (Stanford University) and EP-A-0 194 276 (Celltech Limited).
EP-A-0 194 276 discloses a process for preparing an antibody molecule having the variable domains from a mouse MAb and the constant domains from a human immunoglobulin. It also describes the production of an antibody molecule comprising the variable domains of a mouse MAb, the CH1 and CL domains of a human immunoglobulin and a non-immunoglobulin-derived protein in place of the Fc portion of the human immunoglobulin.
Subsequently, a number of further patent applications have been published relating to chimeric antibodies, including tumour specific chimeric antibodies. Among these applications are WO-A-87/02671 (Int. Gen. Eng. Inc.), EP-A-0 256 654 (Centocor), EP-A-0 266 663 (Int. Gen. Eng. Inc. and Oncogen), WO-A-89/00999 (Int. Gen. Eng. Inc.) and EP-A-0 332 424 (Hybritech Inc.).
Such humanised chimeric antibodies, however, still contain a significant proportion of non-human amino acid sequence, i.e. the complete variable domains. Thus, such humanised antibodies may elicit some HAMA response, particularly if administered over a prolonged period [2].
In an alternative approach, described in EP-A-0 239 400 (Winter), the complementarity determining regions (CDRs) of a mouse MAb have been grafted onto the framework regions of the variable domains of a human immunoglobulin by site directed mutagenesis using long oligonucleotides. Such CDR-grafted humanised antibodies are less likely to give rise to a HAMA response than humanised chimeric antibodies in view of the lower proportion of non-human amino acid sequence which they contain. There are three CDRs (CDR1, CDR2 and CDR3) in each of the heavy and light chain variable domains.
The earliest work on CDR-grafted humanised MAbs was carried out on a MAb recognising the synthetic antigen NP or NIP. However, subsequently, examples in which a mouse MAb recognising lysozyme and a rat MAb recognising an antigen on human T cells respectively were humanised have been described [3, 4]. The preparation of the CDR-grafted antibody to the antigen on human T cells is also described in WO-A-89/07452 (Medical Research Council). Recently the preparation of a humanised CDR-grafted antibody that binds to the interleukin 2 receptor has been described [5]. Further examples of humanised CDR-grated antibodies having specificity for anti-viral [6, 7], anti-tumour [8] and anti-T cell [9 and EP-A-0 403 156] antigens have been described more recently.
Our copending International Patent Specification No. WO-A-91/09967 relates to the CDR grafting of antibodies in general.
It has been widely suggested that immunoglobulins, and in particular MAbs, could potentially be very useful in the diagnosis and treatment of cancer [10, 11]. There has therefore been much activity in trying to produce immunoglobulins or MAbs directed against tumour-specific antigens. So far, over one hundred MAbs directed against a variety of human carcinomas have been used in various aspects of tumour diagnosis or treatment [12].
There have been a number of papers published concerning the production of chimeric monoclonal antibodies recognising cell surface antigens. For instance, genetically engineered murine/human chimeric antibodies which retain specificity for tumour-associated antigens have been described [13 and WO-A-89/01783]. Also, a recombinant murine/human chimeric monoclonal antibody specific for common acute lymphocytic leukaemia antigen has been described [14].
We have now prepared humanised antibodies to human milk fat globule (HMFG) derived from the anti-HMFG mouse MAb CTMO1 [15].
According to the present invention, there is provided a humanised antibody molecule (HAM) having specificity for human milk fat globule (HMFG) and having an antigen binding site wherein at least one of the complementarity determining regions (CDRs) of the variable domain is derived from the mouse monoclonal antibody CTMO1 (CTMO1 MAb) and the remaining immunoglobulin-derived parts of the HAM are derived from a human immunoglobulin or an analogue thereof.
The HAM may comprise a chimeric humanised antibody or a CDR-grafted humanised antibody. When the HAM comprises a CDR-grafted humanised antibody, each heavy or light chain variable domain may comprise only one or two CTMO1-derived CDRs. Preferably, however, all three heavy and light chain CDRs are derived from CTMO1.
The CTMO1 MAb is a mouse MAb of the type IgG1-kappa raised against the membrane-associated antigen of HMFG and has been extensively studied [15]. The CTMO1 MAb has been shown to recognise breast, ovarian and non-small cell lung cancers. It has been shown to internalise rapidly into target cells. Conjugates of CTMO1 and calichaemicin display highly specific cytotoxicity against appropriate cell lines, (see U.S. Pat. No 5,053,394).
High levels of the antigen recognised by the CTMO1 MAb have been detected circulating in the blood of patients suffering from breast cancer. This may have a deleterious effect on pharmacokinetics and tumour localisation in vivo. However, circulating antigen levels in the blood of patients suffering from ovarian cancer are lower than those in breast cancer patients. It is therefore believed that the HAM of the present invention will be of particular use in the treatment of ovarian cancer.
It is believed that the CTMO1 MAb recognises the polymorphic epithelial mucin (PEM) of HMFG. Thus, preferably, the present invention provides a HAM which recognises the PEM of HMFG.
Surprisingly, it has been found that humanising the CTMO1 MAb does not substantially adversely affect its binding activity or internalisation, and can create, particularly by CDR grafting, a HAM which has better binding and internalisation characteristics than the murine antibody (see Table 1 hereinafter). This produces a HAM which is of use in both therapy and diagnosis of certain human carcinomas, for example carcinomas of ovary, breast, uterus and lung.
Preferably, the HAM of the present invention is produced by recombinant DNA technology.
The HAM of the present invention may comprise: a complete antibody molecule, having full length heavy and light chains; a fragment thereof, such as an Fab, Fabxe2x80x2, (Fabxe2x80x2)2 or Fv fragment; a single chain antibody fragment, e.g. a single chain Fv; a light chain or heavy chain monomer or dimer; or a fragment or analogue of any of these or any other molecule with the same specificitiy as the CTMO1 MAb.
The HAM of the present invention may have attached to it an effector molecule such as a cytotoxic or cytostatic agent, or a reporter group, for example an atom or molecule such as a radionuclide, or complexed radionuclide capable of being detected while inside the human body. For instance, the HAM may have a macrocycle, for chelating a heavy metal atom, or a toxin, such a ricin, attached to it by a covalent bridging structure. Alternatively, the procedures of recombinant DNA technology may be used to produce a HAM in which the Fc fragment, CH2 or CH3 domain of a complete molecule has been replaced by or has attached thereto by peptide linkage a functional non-immunoglobulin protein, such as an enzyme or toxin molecule.
The remaining non-CTMO1 immunoglobulin-derived parts of the HAM may be derived from a suitable human immunoglobulin. For instance, when the HAM is a CDR-grafted HAM, appropriate variable region framework sequences may be used having regard to the class or type of the CTMO1 donor antibody from which the antigen binding regions are derived. Preferably, the type of human framework used is of the same or similar class or type as the donor antibody (CTMO1 is IgG1-kappa). Advantageously, the framework is chosen to maximise or optimise homology with the donor antibody sequence, particularly at positions spatially close to or adjacent the CDRs. Examples of human frameworks which may be used to construct CDR-grafted HAMs are LAY, POM, TUR, TEI, KOL, NEWM, REI and EU [16]. KOL and NEWM are suitable for heavy chain construction. REI is suitable for light chain construction. EU is particularly suitable for both heavy chain and light chain construction. Preferably, the EU framework is used as the human framework for both heavy and light chain variable domains in view of its high level of homology with the CTMO1 MAb.
The light or heavy chain variable regions of the HAM may be fused to human light or heavy chain constant domains as appropriate, (the term xe2x80x9cheavy chain constant domainsxe2x80x9d as used herein are to be understood to include hinge regions unless specified otherwise). The human constant domains of the HAM, where present, may be selected having regard to the proposed function of the antibody, in particular the effector functions which may be required. For example, the heavy chain constant domains fused to the heavy chain variable region may be human IgA, IgG or IgM domains. Preferably human IgG domains are used. IgG1 and IgG3 isotype domains may be used when the HAM is intended for therapeutic purposes and antibody effector functions are required. Alternatively, IgG2 and IgG4 isotype domains may be used when the HAM is intended for purposes for which antibody effector functions are not required, e.g. for imaging, diagnostic or cytotoxic targeting purposes. Light chain human constant domains which may be fused to the light chain variable region include human Lambda or, especially, human Kappa chains.
Analogues of human constant domains may alternatively be advantageously used. These include those constant domains containing one or more additional amino acids than the corresponding human domain, or those constant domains wherein one or more existing amino acids of the corresponding human domain has been deleted or altered. Such domains may be obtained, for example, by oligonucleotide directed mutagenesis. In the present invention, a particularly useful analogue of a heavy chain constant domain is an IgG4 constant domain in which a serine residue at position 241 of the corresponding naturally occuring human domain has been altered to a proline residue.
The remainder of the HAM need not comprise only protein sequences from human immunoglobulins. For instance, a gene may be constructed in which a DNA sequence encoding part of a human immunoglobulin chain is fused to a DNA sequence encoding the amino acid sequences of a polypeptide effector or reporter molecule.
According to a second aspect of the present invention, there is provided a process for producing the HAM of the first aspect of the invention, which process comprises:
(a) producing in an expression vector an operon having a DNA sequence which encodes an antibody heavy or light chain comprising a variable domain wherein at least one of the CDRs of the variable domain is derived from the CTMO1 MAb and the remaining immunoglobulin-derived parts of the antibody chain are derived from a human immunoglobulin;
(b) producing in an expression vector an operon having a DNA sequence which encodes a complementary antibody light or heavy chain comprising a variable domain wherein at least one of the CDRs of the variable domain is derived from the CTMO1 MAb and the remaining immunoglobulin-derived parts of the antibody chain are derived from a human immunoglobulin;
(c) transfecting a host cell with both operons; and
(d) culturing the transfected cell line to produce the HAM.
The cell line may be transfected with two vectors, the first vector containing the operon encoding the light chain-derived polypeptide and the second vector containing the operon encoding the heavy chain-derived polypeptide. Preferably, the vectors are identical except in so far as the coding sequences and selectable markers are concerned so as to ensure as far as possible that each polypeptide chain is equally expressed.
Alternatively, a single vector may be used, the vector including the operons encoding both light chain- and heavy chain-derived polypeptides.
In further aspects, the invention also includes DNA sequences coding for the heavy and light chains of the HAM of the present invention, cloning and expression vectors containing these DNA sequences, host cells transformed with these DNA sequences and processes for producing the heavy or light chains and antibody molecules comprising expressing these DNA sequences in a transformed host cell.
The general methods by which the vectors may be constructed, transfection methods and culture methods are well known per se [17, 18].
The DNA sequences which encode the CTMO1 heavy and light chain variable domain amino acid sequences (and the corresponding deduced amino acid sequences) are given hereinafter in the sequence listing as Sequence ID No. 1 and Sequence ID No. 2 respectively.
DNA coding for human immunoglobulin sequences may be obtained in any appropriate way. For example, amino acid sequences of preferred human acceptor frameworks, such as LAY, POM, KOL, REI, EU, TUR, TEI and NEWM, are widely available to workers in the art. Corresponding DNA sequences which code for these amino acid sequences may be inferred or deduced by reverse application of the genetic code. Similarly, the amino acid sequences of human constant region domains are well known and DNA sequences which code for them may be readily deduced.
The standard techniques of molecular biology may be used to prepare DNA sequences coding for CDR-grafted products. Desired DNA sequences may be synthesised completely or in part using oligonucleotide synthesis techniques. Site-directed mutagenesis and polymerase chain reaction (PCR) techniques may be used as appropriate. For example, oligonucleotide directed synthesis [19] may be used. Also, oligonucleotide directed mutagenesis of a pre-existing variable domain region [3, 4] may be used. Enzymatic filling-in of gapped oligonucleotides using T4 DNA polymerase [5] may be used.
Any suitable host cell/vector system may be used for expression of the DNA sequences coding for the chimeric or CDR-grafted heavy and light chains. Bacterial, e.g. E. coli, and other microbial systems may be used, in particular for expression of antibody fragments, e.g. Fv, Fab and Fabxe2x80x2 fragments and single chain antibody fragments, e.g. single chain Fvs. Eucaryotic, e.g. mammalian host cell, expression systems may be used for production of larger chimeric or CDR-grafted antibody products, including complete antibody molecules. Suitable mammalian host cells include CHO cells and myeloma or hybridoma cell lines, for example NSO cells.
The present invention also includes therapeutic and diagnostic compositions containing the HAM of the invention and uses of such compositions in therapy and diagnosis. Such therapeutic and diagnostic compositions typically comprise a HAM according to the invention in combination with a pharmaceutically acceptable excipient, diluent or carrier, e.g. for in vivo use.
Therapeutic and diagnostic uses typically comprise administering an effective amount of a HAM according to the invention to a human subject. The exact dose to be administered will vary according to the intended use of the HAM and on the age and condition of the patient but may be typically varied from about 0.1 mg to 1000 mg, for example from about 1 mg to 500 mg. The HAM may be administered as a single dose, or in a continuous manner over a period of time. Doses may be repeated as appropriate. The HAM may be formulated in accordance with conventional practice for administration by any suitable route, and may generally be in a liquid form [e.g. a solution of the antibody in a sterile physiologically acceptable buffer] for administration by for example an intravenous, intraperitoneal or intramuscular route.
In the HAM of the first aspect of the invention and the process of the second aspect of the invention, the heavy and light chain variable domains of the HAM may comprise either the entire variable domains of the CTMO1 MAb or may comprise framework regions of a human variable domain having grafted thereon one, two or all three of the CDRs of the CTMO1 MAb. Thus, the HAM may comprise a chimeric humanised antibody or a CDR-grafted humanised antibody.
When the HAM is a CDR-grafted humanised antibody, in addition to the CDRs, specific variable region framework residues may be altered to correspond to non-human, i.e. the CTMO1 mouse, residues.
Preferably, the CDR-grafted humanised antibodies of the present invention include CDR-grafted humanised antibodies as defined in our International Patent Specification WO-A-91/09967. The disclosure of WO-A-91/09967 allowed U.S. application Ser. No. 08/303,569, filed Aug. 16, 1991, is incorporated by reference.
Preferably, the CDRs of light chain correspond to the Kabat CTMO1 MAb CDRs at CDR1 (residues 24-34) and CDR2 (residues 50-56) and to the structural loop residues (residues 91-96) or Kabat CTMO1 MAb CDR residues (residues 89-97) in CDR3. (The residue designations given above and elsewhere in the present application are numbered according to the Kabat numbering system [16]). In addition, the light chain may have mouse CTMO1 residues at one or more of residues 1, 2, 3, 36, 37, 45, 48, 49, 60, 63, 70, 84, 85, 87 and 108. In preferred embodiments, when the human framework used is EU, the light chain comprises Kabat CTMO1 MAb CDRs at all of CDR1, CDR2 and CDR3 and preferably additional CTM01 residues at positions 3, 36, 37, 45, 48, 63 and 108, or especially additional CTMO1 residues at positions 3, 36, 63 and 108 only.
Preferably, the CDRs of the heavy chain correspond to the Kabat CTMO1 MAb CDRs at all of CDR1 (26 to 35), CDR2 (50 to 65) and CDR3 (94 to 100). In addition, the heavy chain may have mouse CTMO1 residues at one or more of residues, 2, 6, 23, 37, 48, 49, 67, 69, 73, 76, 78, 80, 88, 91 and 94. In particularly preferred embodiments, when the human framework used is EU, the heavy chain framework comprises additional CTM01 MAb residues at positions 2, 37, 71 and 73, and especially in addition at positions 48, 67 and 69.
In addition, EU has a particularly idiosyncratic J region between residues 103 to 113 and it may be useful to include the murine amino acids, a consensus human J region or a suitable combination of both at residues 103 to 108 inclusive. When the EU framework is used, preferably heavy chain residues 94, 103, 104, 105 and 107 are murine residues, since in the case of these residues, the murine sequence is more frequently found in human VH sequences than the EU residues.